Candida albicans is an opportunistic fungal pathogen of humans. Treatment of C. albicans infections relies on azoles, which target the lanosterol 14α-demethylase (Erg11p) encoded by the ERG11 gene. Our results show that targeted gene disruption of ERG11 can result in resistance to ergosterol-dependent drugs (azoles and amphotericin B), auxotrophy and aerobically viable erg11Δ/Δ cells. Abnormal sterol deposition and lack of ergosterol in the erg11Δ/Δ strain leads to reduced plasma membrane (PM) fluidity, as well as dysfunction of the vacuolar and mitochondrial membranes, resulting respectively in defects in vacuole fusion and a reduced intracellular ATP level. The altered PM structure of the erg11Δ/Δ strain contributes to delocalisation of H+-ATPase and the Cdr1 efflux pump from the PM to vacuoles and, resulting in a decrease in PM potential (Δψ) and increased sensitivity to ergosterol-independent xenobiotics. This new insight into intracellular processes under Erg11p inhibition may lead to a better understanding of the indirect effects of azoles on C. albicans cells and the development of new treatment strategies for resistant infections.
Adherence of the fungus, Candida albicans, to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans.The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain.Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole-gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment. K E Y W O R D SCandida albicans, Cdr1, cell surface hydrophobicity, ergosterol, fluconazole synergism, gentamicin
Recognizing the β-glucan component of the Candida albicans cell wall is a necessary step involved in host immune system recognition. Compounds that result in exposed β-glucan recognizable to the immune system could be valuable antifungal drugs. Antifungal development is especially important because fungi are becoming increasingly drug resistant. This study demonstrates that lipopeptide, surfactin, unmasks β-glucan when the C. albicans cells lack ergosterol. This observation also holds when ergosterol is depleted by fluconazole. Surfactin does not enhance the effects of local chitin accumulation in the presence of fluconazole. Expression of the CHS3 gene, encoding a gene product resulting in 80% of cellular chitin, is downregulated. C. albicans exposure to fluconazole changes the composition and structure of the fungal plasma membrane. At the same time, the fungal cell wall is altered and remodeled in a way that makes the fungi susceptible to surfactin. In silico studies show that surfactin can form a complex with β-glucan. Surfactin forms a less stable complex with chitin, which in combination with lowering chitin synthesis, could be a second anti-fungal mechanism of action of this lipopeptide.
The regulation of telomere and centromere structure and function is essential for maintaining genome integrity. Schizosaccharomyces pombe Rrp1 and Rrp2 are orthologues of Saccharomyces cerevisiae Uls1, a SWI2/SNF2 DNA translocase and SUMO-targeted ubiquitin ligase. Here, we show that Rrp1 or Rrp2 overproduction leads to chromosome instability and growth defects, a reduction in global histone levels and mislocalisation of centromere-specific histone Cnp1. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2, suggesting that Rrp1 and Rrp2 may be involved in modulating nucleosome dynamics. Furthermore, we confirm that Rrp2, but not Rrp1, acts at telomeres, reflecting a previously described interaction between Rrp2 and Top2. In conclusion, we identify roles for Rrp1 and Rrp2 in maintaining centromere function by modulating histone dynamics, contributing to the preservation of genome stability during vegetative cell growth.
STATEMENTSchizosaccharomyces pombe DNA translocases Rrp1 and Rrp2 modulate centromere and telomere maintenance pathways and dysregulation of their activity leads to genome instability. ABSTRACTHomologous recombination (HR) is a DNA repair mechanism that ensures, together with heterochromatin machinery, the proper replication, structure and function of telomeres and centromeres that is essential for the maintenance of genome integrity. Schizosaccharomyces pombe Rrp1 and Rrp2 participate in HR and are orthologues of Saccharomyces cerevisiae Uls1, a SWI2/SNF2 DNA translocase and SUMO-Targeted Ubiquitin Ligase. We show that Rrp1 or Rrp2 upregulation leads to chromosome instability and growth defects. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2. Either Rrp1 or Rrp2 overproduction results in a reduction in global histone levels, suggesting that Rrp1 and Rrp2 may modulate nucleosome dynamics. In addition we show that Rrp2, but not Rrp1, acts at telomeres. We propose that this role depends on the previously described interaction between Rrp2 and Top2. We conclude that Rrp1 and Rrp2 have important roles for centromere and telomere function and maintenance, contributing to the preservation of genome stability during vegetative cell growth. PLoS Genet. 8, e1002985. Choi, E. S., Cheon, Y., Kang, K. and Lee, D. (2017). The Ino80 complex mediates epigenetic centromere propagation via active removal of histone H3. Nat. Commun. 8, 529. . (2009). The role of novel genes rrp1(+) and rrp2(+) in the repair of DNA damage in Schizosaccharomyces pombe. DNA Repair (Amst). 8, 627-636. Involvement of Schizosaccharomyces pombe rrp1+ and rrp2 + in the Srs2-and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition.Allshire, R. C. (1996). Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function.
Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicans erg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicans erg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicans erg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.
Due to the increasing number of Candida albicans’ infections and the resistance of this pathogenic fungus to drugs, new therapeutic strategies are sought. One of such strategies may be the use of static magnetic field (SMF). C. albicans cultures were subjected to static magnetic field of the induction 0.5 T in the presence of fluconazole and amphotericin B. We identified a reduction of C. albicans hyphal length. Also, a statistically significant additional effect on the viability of C. albicans was revealed when SMF was combined with the antimycotic drug amphotericin B. The synergistic effect of this antimycotic and SMF may be due to the fact that amphotericin B binds to ergosterol in plasma membrane and SMF similarly to MF could influence domain orientation in plasma membrane (PM).
Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.
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